Chiriottieditori.it

Antibiotic susceptibility of potentiAlly probiotic LactobaciLLus strAins JunhuA hAna, DAhuAn chena, shAnshAn lic, Xingfeng lia, Wen-Wen Zhoud, bolin ZhAngb,*, yingmin JiAa,*
aCollege of Biological Science and Engineering, Hebei University of Science and Technology,
Shijiazhuang, Heibei, 050018, China
bSchool of Biological Science and Biotechnology, Beijing Forestry University, Beijing, 100083, China
c Dongcheng District Center For Disease Control And Prevention, Beijing, 100009, China
dSchool of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou Zhejiang,
*Corresponding authors:
Yingmin Jia, Tel. +86 311 81668010, [email protected]
Bolin Zhang, Tel. +86 10 62338221,
susceptibility of 29 Lactobacilli to 13 antibiotics was assayed by paper disc diffusion method.
Plasmids and gastrointestinal tolerance were detected. the relationship between plasmids and
antibiotic resistance was discussed. the results showed that all of the strains were resistant to
bacitracin, polymyxin b, kanamycin, and nalidixic acid. Many strains were relatively sensitive to
chloramphenicol and tetracycline. six strainscontained plasmids and showed good gastrointesti-
nal tolerance. β-lactam resistance gene blr was found in the plasmid of L. plantarum cIcc 23180
by Pcr. the study will be helpful to promote the safety evaluation and development of potential-
ly probiotic lactic acid bacteria.
- Keywords: antibiotic resistance; Lactobacillus; gastrointestinal tolerance; plasmid; probiotic -
282Ital. J. Food Sci., vol. 27 - 2015
testinal conditions was investigated. by plas-
mid elimination and Pcr, the relationship be-
tween the plasmid proﬁles and resistance pat-
due to the claimed beneﬁts, Lactobacillus
terns of the strains was explored. this will pro-
bacteria are widely used in food, feed, medical
vide a reference for the safety evaluation meth-
and health related ﬁelds. Many lactic acid bac-
od and also will be helpful to improve the eval-
teria (LAb), such as Streptococcus thermophi-
uation system of probiotics.lus and Lactobacillus delbruekii subsp. bulga-ricus, have been used safely for a long history.
they are agreed to be secure and do not have
2. MAtErIALs And MEtHods
the possibility of pathogenic. currently, new
beneﬁcial bacteria are being developed contin-
2.1 Bacterial strains and cultivation
uously and will enter the market. However, the
security of these new strains has caused great
29 Lactobacillus strains used in the study
concern. Evaluation of antibiotic sensitivity is
were listed in table 1. Lactobacillus strains
an important part of safety assessment.
were cultured in Mrs (de Man, rogosa, and
now, overuse of antibiotics has become a
sharpe) medium at 37°c for 18h under anaer-
serious social problem. this led to the emer-
obic condition.
gence of a large number of antibiotic-resistant
Quality control strain recommended by clin-
strains. once the resistance-related factors are
ical and Laboratory standards Institute (cLsI)
tranferred to other microorganisms, especial-
in the antibiotic sensitivity test was E. coli
ly pathogens via food carrier, it will cause tre-
Atcc25922 purchased from the Institute of Mi-
mendous problems. the evolution of antibiotic-
crobiology, chinese Academy of sciences. the
resistant foodborne pathogens has been widely
E. coli Atcc25922 was activated and cultivat-
reported (tHrELfALL et al., 2000; WALsH et al.,
ed in Lb medium (yeast extract 5 g/L, tryptone
2008; WHItE et al., 2002). Moreover, the resist-
10 g/L, nacl 10 g/L) at 37°c.
ance and resistance-related genes of Bifidobac-terium, Lactobacillus and Pediococcus strains to
2.2 Testing of antibiotic susceptibility
different antibiotics were studied systematical-
ly (HuMMEL et al., 2007; Huys et al., 2004; MA-
13 kinds of antibiotics paper discs were pur-
rIA et al., 2007). the tetM gene transfer of tet-
chased from the national Institute for the con-
racycline resistance in Lactobacillus plantar-
trol of Pharmaceutical and biological Products
um among strains was reported by nIAMH et
(table 2), each piece with a diameter of 6.5 mm.
al. (2010).
the quality was fully complied with the WHo
In this study, 29 Lactobacillus strains isolat-
ed from the food environment with potentially
Antibiotic susceptibility was semi-quantita-
probiotic effects (JIn et al., 2009; LI et al., 2009;
tively determined with K-b method by antibiot-
LIu et al., 2011; sun et al., 2009; ZHAo et al.,
ic paper disc diffusion referring to the cLsI as
2013) were used. these strains were assayed for
described by cHArtErIs et al. (1998a).
susceptibility to 13 antibiotics by agar disc dif-
briefly, 1.0 mL Lactobacillus suspension (ap-
fusion method. furthermore, some strains with
proximately 1.5×108 cfu/mL) was added to
higher resistance were analysed for the presence
sterile petri dish with diameter of 90 mm, and
of plasmids. then, the tolerance of the plasmid-
then mixed with a 15 mL MH (Muller Hinton,
containing strains under simulated gastroin-
MH) agar (beef extract powder 6g/L, casein ac-
table 1 - source of the tested strains for antibiotic susceptibility test.Source (original number)Lactobacillus plantarum
CICCa 23124 (L11), CICC 23131 (B31), CICC 23135 (B37), CICC 22195 (C35),
CICC 23166 (ZJ1), CICC 23138 (C8-1), CICC 23180 (CH8)
Lactobacillus rhamnosus
CICC 23119 (1132), CICC 22175 (LL), ATCCb 7469, CICC 22151 (LK-Mt), CICC 22173 (R11)
Lactobacillus salivarius
CICC 23182 (CH-10)
Lactobacillus acidophilus
CICC 22162 (CH-2)
Lactobacillus casei
CICC 23184 (Y5-2b)
Lactobacillus helveticus Lactobacillus pentosus
CICC 23116 (SN23), CICC 22161 (Lp-4), CICC 22160 (Lp-5), CICC 22159 (Lp-B),
CICC 22156 (Ind-3), CICC 22157 (Lp-A)
Lactobacillus paralimentarius
CICC 22148 (412), CICC 22149 (413)
Lactobacillus delbrueckii
CICC 22153 (LB), CICC 22163 (LC)
Lactobacillus paracasei
CICC 22165 (5M1), CICC 22167 (5M7), CICC 23183 (D-400)
aCICC, China center of industrial culture collection. bATCC, American type culture collection.Ital. J. Food Sci., vol. 27 - 2015 283
table 2 - the content of antibiotic paper discs and criteri-
2.4 Gastrointestinal tolerability test
on for judgement.Antibiotics Content/disc inhibition zone diameter (mm)
In order to explore the application safety
in human, the gastrointestinal tolerability of
those lactic acid bacteria containing the plas-
mids were tested.
for acid tolerance test, Lactobacillus cells
≤14 15-17 ≥18
were harvested by centrifugation at 6000 rpm
μg ≤21 22-28 ≥29
for 15 min, washed twice with 0.01 mol/L Pbs,
pH 7.2 after cultured for 18 h at 37°c in Mrs
μg ≤14 15-17 ≥18
broth, and then suspended in 20 mL sterile sa-
line (0.85%, w/v) adjusted to pH 2.5 with ster-
μg ≤13 14-17 ≥18
ile hydrochloric acid.
for bile tolerance test, the modiﬁed method
chloramphenicol 30
μg ≤12 13-17 ≥18
of LEE et al. (1999) was referred to test bile tol-
μg ≤12 13-14 ≥15
erance. the Lactobacillus cells were centrifuged
(6000 rpm, 15 min) after cultivated for 18 h at
multi-polymyxin B
37°c in Mrs broth and suspended in 20 mL ster-
ile saline (0.85 %, w/v) supplemented with 0.3%
(w/v) bile salts (taurocholate, sigma) at pH 6.8.
cNote: R-Resistant; S-Susceptible; I-Intermediate.
forpepsin and trypsin tolerance test, Lactoba-cillus cells were centrifuged (6000 rpm, 15 min)
after cultivated for 18 h at 37°c in Mrs broth,
then suspended in 20 mL sterile simulated gas-
ids hydrolysate 17.5 g/L, soluble starch, 1.5
tric and pancretic juices. fresh simulated gas-
g/L, agar 17 g/L, pH 7.3±0.1) until the medi-
tric and pancreatic juices were prepared daily
um solidiﬁed. the antibiotic paper discs were
according to charteris et al (1998b). Pepsin (sig-
pasted closely onto the solidiﬁed medium with
ma) was added into the simulated gastric juice
sterile tweezers after 5min at room temper-
with a ﬁnal concentration of 5 mg/mL. then the
ature. three discs were pasted in each dish.
pH was adjusted to 2.5 with sterile hydrochloric
the distance was more than 24 mm of each
acid. trypsin (sigma) was added into the sim-
disc center and more than 15 mm from disc
ulated pancreatic juices with a ﬁnal concentra-
edge to the inner edge of dish. next, the dishes
tion of 10 mg/mL. then pH was adjusted to 8.0
were placed at room temperature for 1.5 h and
with 0.1 M naoH.
then incubated at 37°c. After 24 h, the inhibi-
All of the tolerability detection, the initial bac-
tion zone diameter was measured around the
terial counts were adjusted to about 108 cfu/
antibiotic disc with vernier caliper and record-
mL and were checked by viable count determi-
ed. for one tested strain, each antibiotic disc
nation on Mrs agar. for the tolerance assay,
was done 3 times. the inhibition zone diame-
the bacterial suspensions were incubated and
counted at 37°c for 0,1,2,3,4,5,6 h, respectively.
standard sensitive strain of E. coli Atcc25922
All tests were repeated three times to estimate
was used as the control. the operation was the
the standard error.
same as the above.
the antibiotic susceptibility of the tested
2.5 Detection of antibiotic resistance genes
strains was evaluated according to the cLsI cri-
teria (table 2).
Part of the antibiotic-resistant genes of those
lactic acid bacteria containing both plasmids and
2.3 Plasmid DNA extraction
high tolerance were investigated. the β-lactam
resistance-related gene sequence of blr, ECP-
10 mL of Lactobacillus suspension cultured
1569, nps-1 and the chloromycetin resistance-
overnight was centrifugated at 10000 rpm for
related gene sequence of cmlA, cat, cmlA1 in
5 min. then the precipitation was suspended
plasmids were found in national center for bi-
with 500 μL of lysozyme solution (10 mg/mL).
otechnology Information (ncbI). the primers
the mixture was placed in a water bath for 45
were designed and synthesized by beijing sun-
min at 37°c. then plasmid dnA of Lactobacilli
biotech co., Ltd (table 3).
strains was extracted and puriﬁed with dnA ex-
the Pcr programmes were performed with the
traction and puriﬁcation kit of tiangen biotech
plasmid template of the tested strains accord-
(beijing) co., Ltd. Plasmid dnA was observed
ing to the following procedures: initial heating
by agarose gel electrophoresis.
at 94°c for 4 min was followed by 34 cycles of
Antibiotic susceptibility and plasmid stabil-
the following sequence: 94°c for 30 s, 72°c for
ity were tested after cultivated 30 generations
1 min, and 72°c for 1 min. final extension took
at 37°c in Mrs medium according to the above
place at 72°c for 7 min.
the ampliﬁcation products were separated
284Ital. J. Food Sci., vol. 27 - 2015
table 3 - the primers of the resistance genes in the experiment.Sequence of the primer Annealing temperature
5'-CGTCTTATTGAATTAACAGGTTGG -3'
blr--down
5'-CACGAAGCCATGTTGTGTTC -3'
ECP-1569--up
nps-1--up
nps-1--down 5'-GGCGATACCGCTCAGTTAC-3'
cmlA--down 5'-CATGCCCAAACCTAGAAACGC-3'
cat--down 5'-TGGAAGCCATCACAAACG-3'
cmlA1--up
cmlA1--down 5'-CTACGTTGTGGCGTCAATG-3'
by conventional 1.0% (w/v) agarose gel electro-
ance to 13 kinds of antibiotics. to bacitra-
phoresis (100V, 4°c) in tAE (tris-acetate-Ed-
cin, polymyxin b, kanamycin and nalidixic acid,
tA) buffer and visualised by ethidium bromide
the resistance rate of the 29 tested strains was
staining. the target fragment was recovered
100%. to β-lactam and aminoglycosides, the
and sequenced by taKara biotechnology (dali-
resistance percentage was 20.7%-37.9% and
an, china) co., Ltd. the resistance-related gene
86.2%-100%, respectively. All of the 29 strains
of plasmid was determined by comparison with
were mostly sensitive to chloramphenicol and
Among of the tested antibiotics, the nali-
dixic acid and polymyxin b can inhibite dnA
3. rEsuLts And dIscussIon
synthesis and interfer cell membrane forma-
tion, respectively. the resistance of lactobacil-
3.1 Antibiotic susceptibility
lus to these kinds of antibiotics may be due to
the thicker cell wall of Gram-positive bacteria.
Antibiotic susceptibility of the tested strains
While the tested strains showed different sen-
was evaluated according to the anti-microbi-
sitivity to the antibiotics, such as streptomy-
al drug sensitivity standard of cLsI criteria.
cin, kanamycin, tetracycline, chloramphenicol,
the sensitivity of the tested Lactobacillus to 13
gentamicin with protein synthesis inhibitition
kinds of antibiotics was shown in table 4. the
effect. Most lactobacillus strains showed re-
tested 29 strains were generally resisitant to
sistance to those antibiotics against gram-neg-
ative bacteria, for example, streptomycin, gen-
lidixic acid, and were mostly sensitive to chlo-
tamicin, kanamycin. this was consistent with
ramphenicol and tetracycline. the same species
report of Zhang et al (2007).
of Lactobacillus generally had similar resistance
patterns. but there was species speciﬁcity such
3.2 Plasmid DNA extraction
as the different antibiotic sensitivity in L. plan-
of antibiotics-resistant lactobacillus strains
tarum, L. rhamnosus, and L. pentosus. Moreover,
the antibiotic-resistant level of different strains
16 cIcc strains with relatively strong antibi-
is also different.
otic resistance were screened for plasmid extrac-
Antibiotic resistance of the foodborne lactic
tion. As can be seen from fig. 1, among these
acid bacteria had heen reported in the 1980s.
strains, only cIcc 23180, 22161, 22175, 22157,
the researchers generally believed that the re-
23124, and 22154 contained plasmids.
sistance was a result of the long evolution and
L. plantarum cIcc 23180 showed 6 plasmid
it was generally endogenous resistance and ob-
dnA bands and there is one band greater than
tained resistance (Zeng et al., 2004). so, the re-
23 kb. L. pentosus cIcc 22157 showed two plas-
sistant lactic acid bacteria of natural or isolat-
mid dnA bands of 10 kb and 5 kb, respective-
ed from human intestinal can indirectly reflect
ly. L. rhamnosus cIcc 22175 and L. plantarum
the habitat of used antibiotic.
cIcc 23124 contained respectively 2 and 4 of
It can be seen from table 5, the 29 strains
plasmid dnA bands and both of the two strains
contained a 10 kb plasmid. L. helveticus cIcc
Ital. J. Food Sci., vol. 27 - 2015 285

table 5 - the percentage of the antibiotic resistance of 29
Antibiotics Quantity of resistant strains of resistance (%)
penicillin G 11
multi-polymyxin B 29
chloramphenicol 3
L. casei
22154 showed only one plasmid dnA band of
about 10 kb. L. acidophilus
Lactic acid bacteria generally contain plas-
mids. the plasmid size was usually 1.9 kb-84.8
kb. Most of the plasmid was less than 20 kb
(WAnG and LEE, 1997). In the culture process
from generation to generation, many plasmids
might disappear from the bacterial cell, but most
of the plasmids were stable. In the study, the
plasmids of the above six strains and the anti-
biotic susceptivity showed no changes after cul-
tivated 30 generations.
3.3 Gastrointestinal tolerability
resistance to gastrointestinal stress is very
important for one strain to play the potential
probiotic function (GuGLIELMottI et al., 2007).
If the strains have a high tolerance to gastroin-
testinal stress, it will have the chance to sur-
vive and play the probiotic effects in the gastro-
the tolerance of the selected six strains to low
strains to 13 antibiotics.
fig. 1 - the plasmids in Lactobacillus (1.cIcc 23180, 2.cIcc
22161, 3.cIcc 22175, 4.cIcc 22157, 5.cIcc 23124, 6.cIcc
22154. M. λHindIII marker).286Ital. J. Food Sci., vol. 27 - 2015

fig. 2 - the viable counts of strains ccIcc 22154, 22175, 22161, 22157, 23180 and 23124 in the gastrointestinal environ-
ment after 6 hs at 37°c.
A: pH 2.5; b: 3 mg/mL bile; c: 5mg/mL pepsin; d: 10 mg/mL trypsin.
pH, bile salt, pepsin and trypsin is presented in
the viable counts of the tested six strains still re-
fig. 2. As shown in fig. 2A, the viable counts of
mained at 106 cfu/mL or more after 6 h exposure
L. pentosus cIcc 22161 strain reduced to be-
to 10 mg/mL trypsin solution (pH 8.0).
low 106 cfu/mL after 4 h and 1.32 × 104 cfu/
mL after 6 h. However, the viable numbers of
3.4 Detection of Resistance genes
L. helveticus cIcc 22154, L. pentosus cIcc
22157, L. plantarum cIcc 23124, 23180 and
According to the above results, except strain
L. rhamnosus cIcc 22175 were still more than
L. pentosus cIcc 22161 and L. helveticus cIcc
106 cfu/mL after 6 h in the gastric acid of pH
22154, the tested strains may be able to sur-
2.5. thus, these ﬁve strains showed higher tol-
vive in the simulated gastrointestinal environ-
ment. However, if the above strains contain an-
for bile tolerance, except the L. pentosus cIcc
tibiotics-resistant plasmids, there is the possi-
22161, the viable counts of the other ﬁve strains
bility of resistance transfer to other bacteria, es-
were still more than 106 cfu/mL after 6 h in the
pecially pathogenic bacteria. It will be a poten-
medium containing bile salt (fig. 2b). However,
tial hazard to human health and be a serious so-
the viable cells of L. pentosus cIcc 22161 had
cial problem. so, the plasmid-determined resist-
decreased to 2.0×106 cfu/mL within 3 h. And
ant gene should be checked ﬁrstly before sub-
it declined to only 1.8 × 104 after 6 h.
for pepsin tolerance, among of six strains,
After 0.02% sds combined with heat treat-
the viable cells of L. pentosus cIcc 22161 and
ment of the four strains (cIcc 22175, 22157,
L. helveticus cIcc 22154 decreased signiﬁcant-
23124, 23180), only the plasmids of L. plantar-
ly in 6 h and it is less than 104 cfu/mL and 106
um cIcc 23180 were removed and the resist-
cfu/mL after 6 h exposure to 5 mg/mL pepsin
ance to cephalothin and chloromycetin disap-
solution (pH 2.5), respectively (fig. 2c).
peared simultaneously (unpublished results).
for trypsin tolerance, as can be seen in fig. 2d,
so, the primers of β-lactam resistance-relat-
Ital. J. Food Sci., vol. 27 - 2015 287